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Antiviral Research (v.68, #3)
Hepatitis B virus-neutralizing anti-pre-S1 human antibody fragments from large naïve antibody phage library by Sae-Gwang Park; Yong-Joo Jeong; Yong-Yi Lee; Ik-Jung Kim; Su-Kil Seo; Eui-Joong Kim; Heung-Chae Jung; Jae-Gu Pan; Sung-Jae Park; Yeon-Jae Lee; Ik-Sang Kim; In-Hak Choi (pp. 109-115).
We report the construction of a large nonimmunized human phage antibody library in single-chain variable region fragment (scFv) format, which allowed the selection of antibodies that neutralize hepatitis B virus (HBV) in vitro. We generated 1.1×1010 independent scFv clones using the cDNA of functional variable (V) gene segments of heavy and light chains purified from the peripheral blood mononuclear cells of 50 nonimmunized human donors. Using BIAcore, we selected two clones that recognized pre-S1 and neutralized pre-S1 and HBV binding to Chang liver cells. Clone G10 had the highest affinity ( KD=1.69×10−7M), which was higher than that of clone 1E4 that was generated previously from a heavy chain-shuffled immune library. The off-rates of clones were within 10−3s−1 as determined by BIAcore and were comparable to those of antibodies derived from a normal secondary immune response. In the inhibition assays of pre-S1 and virus binding to Chang liver cells using flow cytometry and the polymerase chain reaction, G10 had better neutralizing activity than 1E4. The new phage library may be a valuable source of antibodies with reasonable affinities to different targets, and the anti-pre-S1 G10 may be a good candidate for immunoprophylaxis against HBV infection.
Keywords: Naïve antibody library; Single-chain variable region fragments; Pre-S1 protein; BIAcore
Polymer-bound 6′ sialyl- N-acetyllactosamine protects mice infected by influenza virus by A.S. Gambaryan; E.Y. Boravleva; T.Y. Matrosovich; M.N. Matrosovich; H.-D. Klenk; E.V. Moiseeva; A.B. Tuzikov; A.A. Chinarev; G.V. Pazynina; N.V. Bovin (pp. 116-123).
To develop a mouse model for testing receptor attachment inhibitors of human influenza viruses, the human clinical virus isolate in MDCK cells A/NIB/23/89M (H1N1) was adapted to mice by serial passaging through mouse lungs. The adaptation enhanced the viral pathogenicity for mice, but preserved the virus receptor binding phenotype, preferential binding to 2–6-linked sialic acid receptors and low affinity for 2–3-linked receptors. Sequencing of the HA gene of the mouse-adapted virus A/NIB/23/89-MA revealed a loss of the glycosylation sites in positions 94 and 163 of HA1 and substitutions 275Asp→Gly in HA1 and 145Asn→Asp in HA2. The four mouse strains tested differed significantly in their sensitivity to A/NIB/23/89-MA with the sensitivity increasing in the order of BALB/cJCitMoise, C57BL/6LacSto, CBA/CaLacSto and A/SnJCitMoise strains. Testing of protective efficacy of the polyacrylamide conjugate bearing Neu5Acα2-6Galβ1-4GlcNAc trisaccharide under conditions of lethal or sublethal virus infection demonstrated a strong protective effect of this preparation. In particular, aerosol treatment of mice with the polymeric attachment inhibitor on 24–110h after infection completely prevented mortality in sensitive animals and lessened disease symptoms in more resistant mouse strains.
Keywords: Influenza virus; Sialylglycopolymer; Antivirals; Mice; Anti-adhesion
Human cytomegalovirus-inhibitory flavonoids: Studies on antiviral activity and mechanism of action by David L. Evers; Chih-Fang Chao; Xin Wang; Zhigang Zhang; Shu-Mei Huong; Eng-Shang Huang (pp. 124-134).
We report antiviral activity against human cytomegalovirus for certain dietary flavonoids and their likely biochemical mechanisms of action. Nine out of ten evaluated flavonoids blocked HCMV replication at concentrations that were significantly lower than those producing cytotoxicity against growing or stationary phase host cells. Baicalein was the most potent inhibitor in this series (IC50=0.4–1.2μM), including positive control ganciclovir. Baicalein and genistein were chosen as model compounds to study the antiviral mechanism(s) of action for this series. Both flavonoids significantly reduced the levels of HCMV early and late proteins, as well as viral DNA synthesis. Baicalein reduced the levels of HCMV immediate-early proteins to nearly background levels while genistein did not. The antiviral effects of genistein, but not baicalein, were fully reversible in cell culture. Pre-incubation of concentrated virus stocks with either flavonoid did not inhibit HCMV replication, suggesting that baicalein did not directly inactivate virus particles. Baicalein functionally blocked epidermal growth factor receptor tyrosine kinase activity and HCMV nuclear translocation, while genistein did not. At 24h post infection HCMV-infected cells treated with genistein continued to express immediate-early proteins and efficiently phosphorylate IE1-72. However, HCMV induction of NF-κB and increases in the levels of cell cycle regulatory proteins—events that are associated with immediate-early protein functioning – were absent. The data suggested that the primary mechanism of action for baicalein may be to block HCMV infection at entry while the primary mechanism of action for genistein may be to block HCMV immediate-early protein functioning.
Keywords: Baicalein; Genistein; Antiviral; Cytomegalovirus; Entry; Immediate-early function
Insights into the genetic basis for natural phenotypic resistance of human rhinoviruses to pleconaril by Rebecca M. Ledford; Marc S. Collett; Daniel C. Pevear (pp. 135-138).
Recent phylogenetic analyses of the deduced amino acid sequence of the major viral capsid protein (VP1) of all human rhinovirus (HRV) serotypes revealed two distinct species within the genus: species A (75 serotypes) and species B (25 serotypes). Pleconaril is a novel capsid inhibitor of HRVs. All 75 species A serotypes and 18 of the 25 species B serotypes are susceptible to inhibition by pleconaril in cell culture. The seven resistant serotypes are HRV-4, -5, -42, -84, -93, -97 and -99. We were interested in understanding the genetic basis for phenotypic resistance to pleconaril among these naturally occurring viruses. We compared the 25 amino acids of VP1 that comprise the drug-binding pocket of susceptible and resistant species B viruses. A consistent difference was observed at two positions: the vast majority of susceptible viruses had tyrosine and valine at VP1 residues 152 and 191, respectively (Y152 and V191); all resistant viruses had phenylalanine and leucine at these positions (F152 and L191). HRV-14, a pleconaril susceptible virus, has a drug-binding pocket amino acid composition that differs from the naturally resistant HRV-5 and HRV-42 only at these two positions. To gain further insight into the role of these specific residues in natural resistance to pleconaril, we substituted the amino acids at these two positions individually and in combination in an infectious clone of HRV-14 and tested the rescued virus for susceptibility to pleconaril and virion stability. The results indicate that substitution of V191 to Leu in HRV-14 has a profound negative impact on drug susceptibility but that full resistance to pleconaril is only seen when combined with Phe at position 152 in a HRV-14 double variant (F152, L191). These data identify L191 in species B HRV as a potentially key residue in conferring significantly reduced susceptibility to pleconaril. These results may be useful in distinguishing naturally occurring viral resistance to pleconaril from treatment-emergent resistance.
Keywords: Pleconaril; Picornavirus; Common cold; Antiviral; Natural phenotypic resistance
Evaluation of dendrimer SPL7013, a lead microbicide candidate against herpes simplex viruses by Edwin Gong; Barry Matthews; Tom McCarthy; Jianhua Chu; George Holan; John Raff; Stephen Sacks (pp. 139-146).
Dendrimers are a novel class of polyanionic macromolecules with broad-spectrum antiviral activities and minimal toxicities. A new generation of amide dendrimer, SPL7013, was evaluated as a lead microbicide candidate against herpes simplex viruses (HSV). The plaque reduction assays showed that the 50% effective concentrations (EC50) determined by pre-treatment of cells were 2.0μg/ml for HSV-1 and 0.5μg/ml for HSV-2. Inhibitory effects were also observed on HSV-infected cells with EC50s of 6.1μg/ml for HSV-1 and 3.8μg/ml for HSV-2. These are the mean values from the test results of six batches of SPL7013. SPL7013 was also shown to be equally potent against HSV drug-resistant strains. SPL7013 completely inhibited viral adsorption to Vero cells at concentrations of higher than 3μg/ml. Analyzed by a LightCycler assay after treatment of HSV-infected cells for 17h, SPL7013 showed strong inhibition of HSV DNA synthesis with EC50s of approximately 6.2 and 2.0μg/ml for HSV-1 and HSV-2, respectively. SPL7013 retained its anti-HSV activity even after treatment at acidic pHs 3.0 and 4.0 for 2h. The presence of 10% human serum proteins did not affect the anti-HSV activity of SPL7013. SPL7013 was not toxic to Vero cells up to the highest concentration tested (10,000μg/ml). Effects on cell proliferation were tested on two epithelial cell lines in both stationary and dividing phases. The 50% cytotoxic concentrations (CC50) in all cases were greater than 10,000μg/ml. Our data indicate that SPL7013 is a promising candidate for development as a vaginal microbicide and a therapeutic agent.
Keywords: Dendrimer SPL7013; Herpes simplex viruses; Antiviral activity; Microbicide
Antiviral therapeutic efficacy of foscarnet in hepatitis B virus infection by Yan-Xing Han; Rong Xue; Wei Zhao; Zhen-Xian Zhou; Jian-Nong Li; Hong-Shan Chen; Xiang-Hong Chen; Yan-Ling Wang; Yu-Huan Li; Yin-Wei Wu; Xue-Fu You; Li-Xun Zhao; Jian-Dong Jiang (pp. 147-153).
Foscarnet (PFA), a viral DNA polymerase inhibitor, is a clinical agent for herpes viruses. The goal of the study was to evaluate the therapeutic efficacy of PFA in hepatitis B virus (HBV) infection. Intravenous infusion of PFA (1g/day) for 4 weeks significantly reduced serum HBeAg ( p<0.01) and HBV DNA copies ( p<0.05) in 31 patients who were diagnosed with active chronic HBV infection (CHB) and had not received antiviral treatment previously. Alanine aminotrans-aminase (ALT), aspartate aminotransaminase (AST) and gamma glutamyl transpeptidase (γ-GT) of the patients declined ( p<0.001, 0.001 and 0.01, respectively). Kidney function (blood creatinine and urea nitrogen) remained unchanged. Another 21 lamivudine-resistant CHB patients with mutations at the tyrosine–methionine–aspartate–aspartate motif (YMDD) displayed a response to PFA similar to that mentioned above, with reductions in HBeAg ( p<0.05), HBV DNA ( p<0.01) and liver enzymes (ALT and AST, p<0.001; γ-GT, p<0.05). Moreover, PFA reduced serum HBeAg ( p<0.01), HBV DNA ( P<0.05), AST ( p<0.05) and ALT ( p<0.02) in a cohort of 13 severe CHB patients with advanced liver damage. PFA was also evaluated in vitro and in vivo. PFA inhibited HBV DNA replication in HBV-transfected human HepG2 cells (2.2.15 cells) with reduced amount of HBV RC-DNA and DS-DNA. In the duck HBV-infected ducklings, PFA reduced viral DNA and duck HBsAg in the serum ( p<0.01 for both).
Keywords: HBV infection; Foscarnet; Antiviral therapy
Protective effect of the acyclic nucleoside phosphonate tenofovir toward human T-cell leukemia/lymphotropic virus type 1 infection of human peripheral blood mononuclear cells in vitro by Emanuela Balestrieri; Maria Teresa Sciortino; Antonio Mastino; Beatrice Macchi (pp. 154-162).
9-( R)-[(2-Phosphonomethoxy)propyls]adenine (tenofovir), is an acyclic nucleoside phosphonate known to inhibit HIV replication in vitro and to reduce viremia in HIV-infected patients. Here we have investigated whether tenofovir is able to protect peripheral blood mononuclear cells (PBMCs) from healthy donors against human T-cell leukemia/lymphotropic virus type 1 (HTLV-1) infection in vitro. PBMCs were pre-treated with tenofovir and infected by exposure to an irradiated cell line chronically harbouring HTLV-1. Measurements of viral DNA, as well as viral gene and protein expression, at 4 weeks after infection, revealed that tenofovir at concentrations of 1μM and higher completely protected PBMCs against HTLV-1; lower concentrations did not fully prevent HTLV-1 infection of the cultures. Nevertheless, in the long term, cell growth of infected PBMCs was inhibited in vitro even by 0.1μM tenofovir. In addition, tenofovir directly inhibited HTLV-1 reverse transcriptase activity, in a cell-free assay that utilizes a crude preparation from HTLV-1 viral particles as a source of the enzyme. The selectivity index of tenofovir for HTLV-1, was about four times higher than that of azidothymidine. Taken together our results strongly encourage further studies to investigate the real impact of tenofovir towards HTLV-1 infection.
Keywords: Acyclic nucleoside phosphonates; Tenofovir; HTLV-1; Reverse transcriptase; Reverse transcriptase inhibitors
A novel inhibitor of respiratory syncytial virus isolated from ethnobotanicals by Joshua O. Ojwang; Yan-Hong Wang; Philip R. Wyde; Nikolaus H. Fischer; Wolfgang Schuehly; James R. Appleman; Soreeta Hinds; Craig D. Shimasaki (pp. 163-172).
A novel low molecular weight compound, CJ 4-16-4, isolated from ethnobotanicals using bioassay-guided fractionation, was found to be a potent inhibitor of respiratory syncytial virus (RSV) in vitro and in vivo. In vitro, a very low micromolar efficacious dose was obtained against at least four of subtype A (RSV-Long, RSV A2, and RSV A6 57754) and one of subtype B (Washington) RSV strains without seeing any significant cytotoxicity to Hep-2, MDCK or Vero cell lines. The drug inhibits growth of RSV in Hep-2 cells maintained in tissue culture at a very low concentration (∼0.07μM) with cell toxicity >400μM (TI>5880). In a cotton rat model of RSV infection, the drug was able to reduce viral titers by ∼1log at dose 12.5 and 25mg/kg/day, and by >2log at 100mg/kg/day. This antiviral activity was specific as influenza A and B and herpes simplex 1 and 2 viruses were not inhibited. The results obtained indicate that CJ 4-16-4 warrants clinical development.
Keywords: Inhibitors; Antivirals; Respiratory syncytial virus; Natural product; Ethnobotanicals